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Mechanisms of Mutation and DNA Repair

Mutations

• Spontaneous mutation: occurs in absence of mutagenic agent

• Rate of mutation: probability of change in DNA sequence during a single generation

• Induced mutation: caused by exposure to mutagenic agent=mutagen

Mutations: Phenotypic Effects

• Mutations can be classified by their phenotypic effects

• Germ-line mutations: affect gametes (inherited)

• Somatic mutations: may affect any type of body cell except gametes

(not inherited)

Mutations

• Conditional mutations: produce phenotypic changes under specific (restrictive) conditions but not others (permissive conditions)

• Temperature-sensitive mutations: conditional mutation whose expression depends on temperature

Mutations: Genotypic Effects

• Mutations can be classified by their effect on gene function

• Loss-of-function (null): totally nonfunctional gene product

• Hypomorphic: reduces level of expression

Mutations: Genotypic Effects

• Hypermorphic: above normal level of expression

• Gain-of-function: many are dominant and may cause expression at an abnormal time or in an abnormal place

Molecular Basis of Mutation

Mutations result from changes in the base sequence of DNA:

• Base substitutions -one pair of of DNA nucleotides is replaced by another pair :

-Transition mutations- a purine is substituted for a purine or a pyrimidine is substituted for a pyrimidine

Molecular Basis of Mutation

-Transversion mutations- a purine replaces a pyrimidine or vice versa

• Base substitutions are point mutations which alter one DNA base pair without adding or deleting any base pairs

• Point mutations may affect gene expression in several ways

Point Mutations

Types of point mutations:• Silent substitutions are base substitutions

which do not alter the amino acid composition of the protein encoded by a gene:

-silent mutations may affect the noncoding portion of a gene or may occur in the coding portion but may not alter codon usage

Point Mutations

• Missense mutations change a single amino acid as a result of a change in codon specification:

-missense mutations can have serious consequences on the biological properties of a protein

- sickle cell anemia results from a single amino acid substitution in hemoglobin which alters its structure

Point Mutations

Point mutations can also alter signals used to regulate gene expression:

• Promoter mutations may block transcription

• Splice site mutations may block splicing or create new splice signals

• Nonsense mutations change a codon to a stop codon which results in a premature termination of translation

Insertions and Deletions

• Insertions add one or more nucleotide pairs to DNA sequence

• Deletions remove one or more nucleotide pairs from DNA sequence

• Insertions or deletions involving a multiple of 3 DNA base pairs = in-frame since they do not alter the reading frame of the genetic code

Insertions and Deletions

• Insertions or deletions which involve a non-multiple of 3 DNA base pairs = frameshift mutations since they alter the codon translation reading frame

• Large deletions may remove genes-no gene product is made

• Insertions can result from gene amplification which can result in the overproduction of gene products

Insertions and Deletions

• Deletion mutations in the dystrophin gene cause muscular dystrophy

• Gene amplifications are often observed in human malignancies

• Insertion and deletion mutations may result from unequal crossing-over during recombination or replication slippage during replication of simple tandem repeat sequences

Transposable Elements• Transposable elements are found in

prokaryotes and eukaryotes

• Transposable elements are called selfish DNA because these elements maintain themselves through replication and transposition

Transposable Elements

• Transposition= movement of genetic elements from

one chromosome location to another• transposase=enzyme which

catalyzes movement of genetic element

Transposable Elements

• Steps in transposition:-transposase binds to terminal inverted

repeat sequence-enzymatic cleavage results in transfer to

different chromosomal site-insertion site is random and involves

duplication of 2-12 base pairs • Transposable elements cause mutations

by inactivating genes at sites of insertion

Transposable Elements

• Reverse transcriptase: enzyme using RNA transcript as a template for a DNA daughter strand

• LTR retrotransposons: long terminal repeats

• Non-LTR retrotransposons: no terminal repeats– LINE and SINE: most abundant transposable elements

in mammalian genomes

Spontaneous Mutations

Lederberg’s replica plating:

• bacterial colonies are transferred to velvet pad and from pad to new plate to test for the frequency of phage resistant colonies in a

• population

Mutation Hot Spots

Mutation hot spots have a higher mutation rate than most DNA:

• Cytosine deamination to uracil is often detected at hot spots

• Sites of cytosine methylation result in deamination which converts 5-methylcytosine to thymine

• Both mutations result in GC to AT transitions

Mutation Hot Spots

• Cytosine deamination can be repaired by DNA uracil glycosylase which recognizes the incorrect GU base pair and removes uracil

• AP endonuclease then removes the ribose sugar

• Single-strand gap is repaired by DNA polymerases and nick is sealed by ligase

Induced Mutations

• Base analogs such as 5-bromouracil may be incorporated into DNA during replication instead of thymine and pairs with guanine resulting in AT to GC transition

• Nucleotide analogs can inhibit DNA replication

Chemical Mutagenesis

• Nitrous acid converts amino groups to keto groups altering the base pairing properties of the bases to produce transition mutations

• Alkylating agents add alkyl groups to bases resulting in transition mutations or depurination = loss of guanine

Radiation Mutagenesis

• Ultraviolet radiation (UV) causes adjacent thymines to become covalently linked = pyrimidine dimers

• Ionizing radiation causes formation of free radicals, highly reactive ions which can damage DNA producing serious mutagenic effects

DNA Repair Mechanisms

• Mismatch Repair consists of the excision of a segment of DNA that contains a base mismatch followed by repair

synthesis• Photoreactivation repairs UV- induced pyrimidine

dimers by breaking the covalent linkage between the thymine bases

Excision Repair

• Excision repair is a multistep process in which a segment of damaged DNA is removed and replaced by Resynthesis using the undamaged strand as a template

DNA Repair Mechanisms

• Postreplication repair involves replication of damaged DNA strand which results in a gap at the damaged DNA site

• A segment of the template DNA from the other strand is inserted to repair the gap by recombination

• The gap in the template is repaired